Lecture 13: Autophagy

Question 1

What is autophagy?

Answer 1

• Removal of protein aggregates, old n damaged organelles n invading microbes
• Can be used in developmental remodeling, providing AA, nucleotides, lipids n sugars under low nutrient conditions

Question 2

What is the difference between microautophagy and macroautophagy?

Answer 2

• Microautophagy: direct targeting into a lysosome
  
  • Chaperone-mediated autophagy: entry via a membrane channel
    ○ Hsc70 deliver certain subset of proteins directly thru the lysosome via channel
    § Protein is degraded inside lysosome
  • Macro autophagy: most well-characterized form
    ○ Phagophore captures cargo -> forms autophagosome (vesicle) -> fuses w late endosome, forming amphisome -> fuses w lyososome, forming autolysosme

Question 3

How to tell the difference b/w autophagosome n amphisome in microscopy?

Answer 3

○ Autophagosome: tubular element (ER), double membraned
○ Amphisome: vesicles

Question 4

Describe anterograde traffic from the ER

Answer 4

• Number of destinations can be accessed by the transport network, including secreted vesicles that take products out to the cell surface or into early endosome system
  
  • Early endosome (pH: 6.5) can access recycling compartment or from TGN can go directly into LE (pH 5.5)

Question 5

Describe retrograde traffic from the cell surface

Answer 5

• Via endocytosis
  
  • Primary destination is the early endosome
  • Access the late endosome by acidification
    Access TGN all the way back to the ER

Question 6

Describe organelle fusion n regeneration

Answer 6

• To get to lysosome, you need lysosome late endosome fusion -> forms endolysosome
  
  • How does a protein directed into the ER get into the lysosome?
    ○ ER associated expression of the protein
    § Folding, inactive
    ○ Transported to trans golgi Network, still inactive
    ○ Directed into LE, reduction in pH activates
    ○ Delivers to lysosome by fusion -> forms end lysosome
    ○ Endolysosome starts to tubulate
    § These tubules are removed to form lysosomes

Question 7

How is a low lysosomal pH maintained?

Answer 7

• Pumping V-type ATPases embedded into lysosomal membrane
  
  • Hungry for ATP
  • Convert energy from ATP hydrolysis into movement of hydrogen ions from oxides into lumen of the lysosome
  • As H conc rises -> pH falls
  • PROBLEM: can’t maintain system bc you’re continually driving hydrogen ions against conc gradient you set up
    ○ SOLUTION: counterion may be either a cation (positive) moving out of lysosome or anion (negative) moving into lysosome
    ○ Multiple ion channels that move Ca n Cl ions across the lysosomal membrane -> maintaining pH
    Low pH is what activates lysosomal enzymes including proteases (e.g. nucleases, glycosidases, lipases, phospholipases, phosphates)

Question 8

What is chaperone-mediated autophagy (CMA)?

Answer 8

• Selective lysosomal degradation of proteins bearing Hsc70-binding KFERQ motifs via membrane channel formed from lysosome-associated membrane protein type 2A (LAMP 2A)
  
  • LAMP proteins
    ○ One gene, 3 proteins w different TM n C termini [alternative splicing]
    ○ LAMP-2A: receptor n channel for CMA
    ○ LAMP-2B: required for fusion of autophagosomes w late endosomes/lysosomes
    ○ LAMP-2C: autophagy of nucleic acids
  • KFERQ has 2 positively charged residues (K,R)
    ○ 2 hydrophobic residues (I,F,LV)
    ○ Single negatively charged residue (E,D)
    ○ Single Q that can be either the N or C terminus
    ○ KFER can be in any order

Question 9

Describe the down regulation of CMA

Answer 9

• mTORC2 complex activates Akt1 by phosphorylation
  
  • Activated Akt1 phosphorylates GFAP (glial fibrillary acidic protein)
  • Phosphorylated GFAP inhibits CMA by keeping LAMP 2A inactive
    RESULT: reduced substrate docking

Question 10

What can stimulate mTORC2 activity n inhibit CMA?

Answer 10

• Nutrient rich diets
  
  • High fat content
  • Increasing age

Question 11

Describe the up-regulation of CMA

Answer 11

• Inhibition of mTORC2 n Akt1 -> reduces phosphorylation of GFAP
  
  • Non-phosphorylated GFAP stabilizes multimerizes LAMP 2A
  • RESULT: increased substrate docking
  • Small molecule inhibitors: potential pharmacological intervention
  • CMA is responsive to
    ○ Withdrawal of GF
    ○ Starvation
    ○ Oxidative stress
    ○ DNA damage
    ○ Hypoxia

Question 12

What are some CMA links w health n disease?

Answer 12

• Healthy cells
  ○ Reduces malignant transformation
  
  • Cancer cells
    ○ Degrades
    § Tumour supressors
    § Pro-apoptopic proteins
    ○ Protects against radiation n hypoxia
    ○ Favours growth n metastasis
  • Healthy individuals
    ○ α-SYN
    ○ PARK7
    ○ Tau
  • Neurodegenerative patients
    ○ CMA dysfunction
    § mRNA maturation
    § LAMP2 promoter variants
    § Tau accumulates
    ○ CMA toxicity
    § Mutants of α-SYN
    § PTM PARK7
    § Tau variants

Question 13

Describe how micro autophagy occurs

Answer 13

• Autophagic cargoes are taken up by late endosomes n lysosomes
  
  • Some micro autophagy substrates hv KFERQ motifs n are delivered by Hsc70 by direct binding of the chaperone to phosphatidylserine in the LE membrane
    Autophagic cargos are then degraded in the end lysosomal or lysosomal lumen

Question 14

What is microautophagy, and how does it differ from chaperone-mediated autophagy?

Question 15

How are autophagosomes formed?

Answer 15

• Initiation
  
  • Nucleation
  • Growth
    Closure

Answer 16

• mTORC complexes at the lysosomal membrane
  
  • mTORC: mammalian/mechanistic target of rapamycin complex
    ○ They are nutrient/energy/redox sensors n controlelrs of protein synthesis
    ○ mTORC is a kinase
  • mTORC2 activates mTORC1 via Akt1
  • mTORC1 has a raptor protein while mTORC2 has a rictor protein
  • mTORC1 phosphorylates 2 members of the ULK complex
  • Phosphorylated ULK complexes are inactive n inhibits macroautophagy
    Activation of mTORC reduces both chaperone mediated n macroautophagy